Melanoma is the most rapidly increasing malignancy in the United States. While it accounts for only 4% of all skin cancers, it is responsible for nearly 80% of all skin cancer deaths. If detected early, the disease is easily treated;however, once the disease has spread to distant sites it does not generally respond to adjuvant therapies and the five-year survival is less than 20%. There is a growing need to understand the genetic complexity and cellular signaling heterogeneity of melanoma as treatments move into the era of targeted therapy. Understanding the genetic and signaling profiles that result in tumor formation and maintenance should allow for the identification of appropriate molecular targets and selection of patient populations most likely to respond to specific inhibitors. To better understand melanoma formation, progression, and metastasis we have developed a somatic cell gene delivery mouse model of melanoma. This system allows for the identification of genes altered in this disease in addition to the rapid validation of mutant genes identified in human melanoma samples. The major advantage of this system is the ability to model the multi-step process of carcinogenesis through the introduction of multiple oncogenes into the same cells in vivo without the expense associated with mating multiple strains of mice. In this competitive revision, we propose to expand the scope of the specific aims of the current grant to define the putative genetic alteration(s) that mediate the in vivo growth of the tumors that develop after a long latency and to define the differences that lead to a decreased latency of the in vivo passaged tumor cells compared to the parent cell lines. To this end, we have collected tissue samples and established cell lines for these tumors. Due to the loss of the Cdkn2a locus in these tumors, we suspected that they are genomically unstable. We have already characterized two of the established cell lines for aneuploidy by analysis of DNA content by flow cytometry and found that greater than 50% of the cells were aneuploid. These results will be validated by analysis of chromosomes through metaphase spreads. Copy number changes will be detected at high resolution using array comparative genomic hybridization (aCGH). Specific genes will be investigated using Western blot analysis and fluorescent in situ hybridization (FISH) if gains and/or losses are detected. The presence of mutations will be assessed by direct sequencing. All detected alterations will be validated for their role in tumor progression by both RNA interference (RNAi) and overexpression. We will also perform expression profiling on the tumor samples and validate any significant changes. Because we already possess the samples necessary for this analysis, the studies can be completed within the time frame proposed. Our long-term goals are to translate the knowledge gained from these studies into improvements in molecular targeted therapies for the treatment of advanced melanoma. Public Health Relevance: The increasing incidence of melanoma, in particular among young to middle-aged adults, is a significant public health problem. The long-term goal of this research is to use a novel mouse model of melanoma to identify key proteins required for melanoma formation and growth that could serve as potential targets for the development of more effective drugs for the treatment of advanced stages of this disease.